Further studies on the isolation, characterization and structure of violacein: Purple pigment of Chromobacterium violaceum

The goal of this research was to develop a method for the isolation of violacein which would produce the pigment in its natural state as much as possible, without heat, acid, base or chemical derivations which might generate artifacts; identify an instrumental technique compatible with TLC to determine and monitor the purity of the isolated pigment; and to determine the structure of the purified pigment using modern spectroscopic methods of analysis.; Chemical tools, including chromatographic and spectroscopic techniques, have been used to investigate the properties of violacein and to characterize violacein isolated from the fermentation broth of Chromobacterium violaceum.; The purity of the violacein isolated from the fermentation broth was monitored with FTIR difference spectroscopy. The fractionated violacein used for NMR analysis was obtained from Silica G{dollar}sb{lcub}60{rcub}{dollar} F{dollar}sb{lcub}254{rcub}{dollar}-packed columns.; The solvents used to obtain the most homogeneous fractions included hexane, chloroform, and methanol.; Further fractionation was obtained after the column-isolated fraction was dissolved in methanol. A precipitate formed in the methanol solution which is soluble in ethyl acetate.; The methanol-soluble violacein was termed violacein A and the ethyl acetate-soluble violacein was termed violacein B.; While the two violaceins are chromatographically homogeneous, FTIR analysis of violacein A and violacein B confirmed that the two violaceins are spectroscopically distinguishable.; Low-resolution mass spectrometry and high-resolution electron impact mass spectrometry provided evidence for a molecular weight of 438 amu for violacein A and violacein B. The low-resolution mass spectra for the two violaceins also show two different fragmentation patterns.; High-resolution electron impact mass spectrometry indicates a molecular weight of 438 amu and suggested five possible formulae for violacein A.; The 500 MHz proton NMR analysis of violacein A seems to support the empirical formula of C{dollar}sb{lcub}27{rcub}{dollar}H{dollar}sb{lcub}26{rcub}{dollar}N{dollar}sb3{dollar}O{dollar}sb2{dollar} for violacein rather than the empirical formula of C{dollar}sb{lcub}20{rcub}{dollar}H{dollar}sb{lcub}13{rcub}{dollar}N{dollar}sb3{dollar}O{dollar}sb3{dollar} proposed by earlier workers.; A revised structure of violacein is presented, and based on this structure it is proposed that violacein may function as an integral part of the cell-associated endo-toxins found in Chromobacterium violaceum.